This invention relates to the sequencing of fluorescence labeled DNA and the detecting of the DNA after irradiation by light from a laser, and suitable labels therefor.
In one class of techniques for sequencing DNA, identical strands of DNA are marked with a fluorescent dye. The strands are marked by attaching specially synthesized fluorescent oligonucleotide primers or probes to the strands of DNA, or by attaching the fluorescent dye directly to the strands.
The strands are separated into four aliquots. The strands in a given aliquot are either individually cleaved at or synthesized to any base belonging to only one of the four base types, which are adenine, guanine, cytosine, and thymine (hereinafter A, G, C and T). The adenine, guanine, cytosine, and thymine terminated strands are then electrophoresed for separation and the separated strands are irradiated by a laser and the emission from the fluorescent dye detected. The rate of electrophoresis indicates the DNA sequence.
In a prior art sequencing technique of this class, the fluorescent dyes used as markers have their maximum emission spectra in the visible range, the DNA is subject to irradiation in the visible spectra, and visible spectra detectors and light sources are used. Generally photomultipliers tubes (PMT) are used for detection.
The prior art techniques for DNA sequencing have several disadvantages such as: (1) because the dyes have their emission spectra in the visible region of light spectrum, the lasers used to excite the fluorescent markers, and under some circumstances, the detectors for the light tend to be expensive; and (2) they are relatively noisy due to the background interferences by biomolecules.
Cyanine dyes are known to absorb far-red (600-700 nm) and near-infrared (700-1200 nm) light and techniques for the synthesis of derivatives of the cyanine dyes are known. However, obtaining chromophores with absorbance and emission bands that reduce the effect of background noise during gel electrophoresis apparatus when irradiating with a diode laser scanning has been difficult. Other dyes, not described herein, exhibit absorbance in wavelengths greater than 1200 nm (infrared) and would also provide discrimination against background noise.
Suitable types of cyanine dyes include heptamethine cyanine dyes. Cyanine dyes have traditionally been synthesized by a condensation reaction between a heterocyclic base containing an activated methyl group and an unsaturated bisaldehyde or its equivalent, usually as Schiff base in the presence of a catalyst. Sodium acetate has most frequently been used as a catalyst. In addition to ethanol, solvents such as acetic acid and/or acetic anhydride have also been commonly used as in the synthesis of heptamethine pyrylium dyes. This procedure suffers from several disadvantages, such as for example: (1) the purification of the product is very difficult because of the side products due to aniline; (2) the use of a catalyst interferes with the purity of the product and warrants repeated purification; (3) the reaction is generally faster and cannot be employed for the synthesis of nonsymmetric dyes in one pot; and (4) the scaling up of the reaction products to larger gram quantities leads to several additional problems resulting in poor quality and yield.